The objective of this two-year project is to improve the outcome following heart transplantation by clarifying the mechanisms involved with the induction of donor-specific tolerance via the mixed chimerism approach. Tolerance of kidney allografts in non-human primates (NHPs) has already been achieved in our laboratories using a combination of non-myeloablative conditioning and donor bone marrow transplantation (DBMT) that results in transient mixed hematopoietic chimerism. Based upon these seminal observations, a similar protocol has been tested in human recipients of living related renal allografts and has proven successful. Dramatic as this achievement is, however, it utilizes a therapeutic protocol whose current success is limited to living donor transplantation (conditioning begins on day -6). There is an obvious need to build upon the impressive successes already achieved with living donor kidneys and optimize the mixed chimerism strategy so that it can be applied more widely to cadaveric donor organs like hearts. Recently, we have developed, in our NHP renal allograft model, a novel regimen, the "Delayed Tolerance" protocol which can extend the clinical applicability of the mixed chimerism approach. In this protocol, recipients initially undergo transplantation with conventional immunosuppression and then receive non-myeloablative conditioning and DBMT sometime later. The goal of this project is to develop the Delayed Tolerance regimen using new agents and novel strategies to induce tolerance of cardiac allografts. We will test the hypothesis that either enhancing peripheral mechanisms of tolerance by promoting Tregs or achieving more durable multilineage donor chimerism will result in long-term tolerance of cardiac allografts in NHPs. During the 2-year study we will specifically: (1) Develop a mixed chimerism protocol of Delayed Tolerance induction applicable to human heart transplant recipients, and, (2) Evaluate the role of Tregs in contributing to long-term tolerance of hearts despite the transience of the mixed chimerism induced by the Delayed Tolerance protocol. In fact, this approach could prove to be even more effective than our standard (non-delayed) protocol, since the tolerance conditioning regimen would be instituted in the absence of the proinflammatory cytokines that are invariably present during the peri-transplant period. The detailed mechanistic studies that will be performed and the analyses planned should provide relevant information for designing subsequent studies that will rationally extend the applicability of this recent clinical innovation to increasing numbers of transplant recipients.